Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults and accounts for 20% of all community-acquired pneumonia. This cell wall-less prokaryote moves by gliding motility, which we contend facilitates colonization of the conducting airways of the respiratory tract. Gliding motility is poorly understood, and no homologs of known motility genes, gliding or otherwise, are found in M. pneumoniae. In the current project period we identified a diverse set of gliding- associated genes, demonstrated conclusively that the terminal organelle alone is the gliding motor, defined the requirement and function of several terminal organelle components in gliding, and generated direct evidence that gliding is required for colonization of mucosal epithelium. The studies proposed here build upon that foundation to define further the role of gliding in pathogenesis and explore the mechanical basis for gliding, encompassing three specific aims. Aim 1 addresses the gliding mechanism, focusing on gliding-associated proteins P65, P41 and P24. The terminal organelle detaches from the cell body with the loss of P41 but retains gliding function. Insertions in the P65 gene result in the dragging of surface adhesin P30 from the terminal organelle to the trailing end, where it detaches to leave a trail behind the gliding cell. In the absence of P41, P24 foci appear to move along the long axis of the cell. In the next project period we will apply biochemical and cell imaging approaches including electron cryotomography to explore further the roles of P24, P41, P65, and other selected proteins in gliding. In Aim 2 we will analyze in detail certain gliding mutants exhibiting a distinctive lawn-like growth as a result of disruption of the genes for the only annotated M. pneumoniae protein phosphatase and cognate ser/thr protein kinase. We will confirm cause and effect for these mutants and explore the impact of loss of protine kinase or phosphatase function on the phosphorylation of terminal organelle proteins HMW1 and HMW2. We will also examine cell behavior of these mutants in detail by microcinematography to establish how lawn-like growth is achieved. In Aim 3 we will use a differentiated normal human bronchial epithelium model and wild-type and gliding-defective mycoplasmas to explore how gliding motility specifically contributes to resistance of mucociliary defenses in the colonization of conducting airways. Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults and accounts for 20% of all community-acquired pneumonia. Most infections result in respiratory disease that is chronic and protracted, impacting attendance at school and productivity in the workplace, and permanent lung damage can result. In addition, a growing body of evidence supports a significant, contributing role for M. pneumoniae in onset and recurrence of asthma. The studies proposed here will elucidate the mechanism and role of gliding motility in the colonization of the conducting airways. [unreadable] [unreadable] [unreadable]